The signal transducers and activators of transcription (STATs) are key signal transduction proteins that play a dual role of transducing biological information from cell surface receptors to the cytoplasm and translocating to the nucleus where, as transcription factors, they regulate gene expression (reviewed in Stark, G. R. et al. Annu. Rev. Biochem., 1998, 67:227-264; Horvath, C. M. and J. E. Darnell Curr. Opin. Cell. Biol., 1997, 9:233-239, Ihle, J. N. and I. M. Kerr Trends Genet., 1995, 11:69-74; Schindler, C. and J. E. Darnell Annu. Rev. Biochem., 1995, 64:621-651). Mammalian cells express seven different STATs (1, 2, 3, 4, 5a, 5b, and 6). Gene knockout and other experiments implicated STATs in many important physiological functions such as immune modulation, inflammation, proliferation, differentiation, development, cell survival and apoptosis (Stark, G. R. et al. Annu. Rev. Biochem., 1998, 67:227-264; Horvath, C. M. and J. E. Darnell Curr. Opin. Cell Biol., 1997, 9:233-239, Ihle, J. N. and I. M. Kerr Trends Genet., 1995, 11:69-74; Schindler, C. and J. E. Darnell Annu. Rev. Biochem., 1995, 64:621-651).
STAT tyrosine phosphorylation is required for their biological function. This occurs when cytokines such as interleukin-6 and interferon or growth factors such as PDGF and EGF bind their respective receptors which results in STAT protein recruitment to the inner surface of the plasma membrane in the vicinity of the cytoplasmic portion of the receptors (Ihle, J. N. et al. Annu. Rev. Immunol., 1995, 13:369-398; Leaman, D. W. et al. Faseb J., 1996, 10:1578-1588). Tyrosine kinases that are known to phosphorylate STATs are non-receptor tyrosine kinases such as Src and the Janus kinases, JAK1 and JAK2. Other possible tyrosine kinases that can phosphorylate STATs are peptide growth factor receptors such as PDGFR and EGFR. The cellular levels of STATs that are tyrosine phosphorylated could also be regulated by phosphotyrosine STAT phosphatases such as SHP-1 and SHP-2 (Schaper, F. et al. Biochem J., 1998, 335:557-565; Stofega, M. R. et al. J. Biol. Chem., 1998, 273:7112-7117; Yu, C. L. et al. J. Biol. Chem., 2000, 275:599-604). Once tyrosine phosphorylated, STAT monomers dimerize via reciprocal phosphotyrosine-SH2 interactions and translocate to the nucleus where they bind DNA and regulate gene transcription (Ihle, J. N. and I. M. Kerr Trends Genet., 1995, 11:69-74; Seidel, H. M. et al. Proc. Natl. Acad. Sci. USA, 1995, 92:3041-3045). Whereas tyrosine phosphorylation of STATs regulates dimerization, nuclear translocation and DNA-binding, serine/threonine phosphorylation is believed to regulate the transcriptional activity of STATs (Turkson, J. et al. Mol. Cell Biol., 1999, 19:7519-7528).
Several lines of evidence have implicated some STAT family members in malignant transformation and tumor cell survival (Bowman, T. et al. Cancer Control, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000, 19:6613-6626). STAT3 involvement in oncogenesis is the most thoroughly characterized. First, STAT3 is found constitutively tyrosine phosphorylated and activated in many human cancers (Bowman, T. et al. Cancer Control, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000, 19:6613-6626; Bowman, T. et al. Oncogene, 2000, 19:2474-2488). This abnormal activation of STAT3 is prevalent in breast, pancreas, ovarian, head and neck, brain, and prostate carcinomas as well as melanomas, leukemias and lymphomas. In those tumors investigated, aberrant STAT3 activation is required for growth and survival (Bowman, T. et al. Cancer Control, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000, 19:6613-6626; Bowman, T. et al. Oncogene, 2000, 19:2474-2488). Second, many known oncogenes, especially those belonging to the non-receptor tyrosine kinase family such as src, induce constitutive activation of STAT3 (Yu, C. L. et al. Science, 1995, 269:81-83). Third, expression of a constitutively-activated mutant of STAT3, where stable dimerization was forced through disulfide covalent linkage, was shown to be sufficient to induce cell transformation and tumor growth in nude mice (Bromberg, J. F. et al. Cell, 1999, 98:295-303). Finally, perhaps the most compelling evidence for the requirement of STAT3 for oncogenesis and its validation as an anticancer drug target comes from experiments where a dominant negative form of STAT3 was used in cultured cells as well as in gene therapy animal experiments to show that blocking aberrant activation of STAT3 results in inhibition of tumor growth and survival and induction of apoptosis with little side effects to normal cells (Niu, G. et al. Cancer Res., 1999, 59:5059-5063; Catlett-Falcone, R. et al. Immunity, 1999, 10:105-115).
Much of modern anticancer drug discovery approaches have focused on targeting signal transduction pathways involving receptor tyrosine kinases (e.g., ErbB2, EGFR), farnesylated proteins (e.g., Ras), and non-receptor cytosolic kinases (e.g. Raf, Mek, PI3K, and Akt) (Sebti, S. New Drug Targets and Therapies for Cancer, 2000, 6549-6692). These important efforts resulted in several novel agents such as RTK monoclonal antibodies and RTK, farnesyltransferase, Raf, and Mek inhibitors that are presently in clinical trials, such as the Bcr-Abl tyrosine kinase inhibitor STI-571 (GLEEVEC), which has recently been approved by the FDA for chronic myelogenous leukemia.
In contrast to the heavily exploited area described above, little has been done to target the STAT3 signaling pathway. Yet, experiments in animal models using gene therapy with a dominant negative form of STAT3 and a constitutively-active mutant of STAT3, as well as the prevalence of constitutively-activated STAT3 in many human cancers, strongly suggest that STAT3 has a causal role in oncogenesis (Bowman, T. et al. Cancer Control, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000, 19:6613-6626; Bowman, T. et al. Oncogene, 2000, 19:2474-2488). Furthermore, the fact that constitutive activation of STAT3 induces genes such as cyclin D1, c-myc, and bcl-xl that are intimately involved in oncogenesis and tumor survival, coupled with the fact that constitutively-activated STAT3 is required for survival of some human cancer cells, further validates the STAT3 signaling pathway as a selective cancer drug discovery target (Bowman, T. et al. Cancer Control, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000, 19:6613-6626; Bowman, T. et al. Oncogene, 2000, 19:2474-2488.
Based on the observations described above, some researchers have undertaken to target STAT3 for the development of novel anti-cancer drugs (reviewed in Bowman, T. et al. Cancer Control, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000, 19:6613-6626; and Bowman, T. et al. Oncogene, 2000, 19:2474-2488). Depending on the aberrant genetic alterations that result in constitutively tyrosine-phosphorylated, activated STAT3, several approaches can be undertaken including blocking ligand/receptor interactions, inhibiting receptor and non-receptor tyrosine kinases, activating phosphotyrosine STAT3 phosphatases, and blocking STAT3 dimerization, nuclear translocation, DNA-binding, and gene transcription. In addition, gene therapy, anti-sense, or RNAi approaches can also be attempted.
JSI-124 is a plant natural product previously identified as cucurbitacin I, a member of the cucurbitacin family of compounds that are isolated from various plant families, such as the Cucurbitaceae and Cruciferae, and that have been used as folk medicines for centuries in countries such as China and India. However, little was known about the biological activities of the various cucurbitacins until recently. Some cucurbitacins have been shown to have anti-inflammatory and analgesic as well as cytotoxic effects. Furthermore, cucurbitacins were also found to inhibit DNA, RNA and protein synthesis in HeLa cells (Witkowski, A. et al. Biochem Pharmacol, 1984, 33:995-1004) and inhibit proliferation of HeLa cells (Witkowski, A. et al. Biochem Pharmacol, 1984, 33:995-1004), endothelial cells (Duncan, M. D. and K. L. Duncan J. Surg. Res., 1997, 69:55-60) and T-lymphocytes (Smit, H. F. et al. J. Nat. Prod., 2000, 63:1300-1302). Finally, some cucurbitacins were shown to suppress skin carcinogenesis (Konoshima, T. et al. Biol. Pharm. Bull., 1995, 18:284-287), inhibit cell adhesion (Musza, L. L. et al. J. Nat. Prod., 1994, 57:1498-1502) and disrupt the actin and vimentin cytoskeleton in prostate carcinoma cells (Duncan, K. L. et al. Biochem Pharmacol, 1996, 52:1553-1560; Duncan, M. D. and K. L. Duncan J. Surg. Res., 1997, 69:55-60).